Separation and Further Characterization of Hematopoietic Cell Populations Based on Phenotypic and Biophysical Properties

University dissertation from Division of Hematology and Transfusion Medicine, Department of Laboratory Medicine, Lund University

Abstract: Hematopoietic stem cells (HSC) are multipotent and possess self-renewal capacity. Thus, they can sustain a life-long hematopoiesis and are also capable of restoring the hematopoietic system when transplanted to a patient. In allogeneic hematopoietic stem cell transplantation (HSCT), a potent graft-versus-leukemia (GvL) effect must be balanced against the concomitant risk of severe immunological response to non-malignant host cells; graft-versus-host-disease (GvHD). In case of recipient-donor tissue antigen disparity, such as human leukocyte antigen (HLA)-mismatch or ABO-incompatibility, depletion of allo-reactive or incompatible blood constituents by HSCT graft processing may be required. The aim of this work was to investigate the development of human erythroid/myeloid progenitor cells and their progeny in normal differentiation and in the chimeric state of transfusion and allogeneic HSCT. Furthermore, novel technology platforms for optimal HSCT graft engineering in the setting of recipient-donor tissue antigen disparity were investigated.
We utilized two in vitro culture systems (Paper II) to obtain cells of advanced neutrophil and erythroid maturation from adult human CD34+ bone marrow cells, and to study the relationship between clonogenicity, gene expression and phenotype during early myeloid development. Cells were sorted by fluorescence-activated-cell-sorting (FACS) according to selected surface markers into defined populations of sequential developmental stages for further analysis. Thus the expression profiles of several genes could to be directly correlated to changes in clonogenic potential and to lineage commitment, as defined by surface expression of CD15 (neutrophil differentiation) and group A antigen (eryhtroid differentiation).
With regard to the established surface markers for neutrofil and erythroid lineage-affiliation, we investigated (Paper I) the origin of reappearing recipient-derived RBC in a patient with relapsed myelodysplastic syndrome (MDS) following allogeneic HSCT. The presence of the original cytogenetic 20q-deletion in myeloid cell populations of different maturity, suggested an origin common to the original myeloid malignant clone and the reappearing autologous RBC, i.e. a progenitor cell with preserved granulocyte/monocyte and megakaryocyte/erythroid potential. We continued our study of chimeric post-HSCT recipients by addressing (paper V) the phenomenon of blood group A/B antigen acquisition by donor group O RBC, following transfusion or ABO-incompatible HSCT. Using a highly sensitive flow cytometry assay, donor group O RBC were found to express variable levels of acquired antigen, ranging from very small amounts in non-secretor individuals to ABO-subgroup levels in group A1 secretors. Our findings support the major role of A/B antigen adsorption from secretor plasma, but indicate a secretor-independent mechanism for A/B-antigen acquisition.
To improve graft processing in HLA-mismatched HSCT, we investigated (Paper III) and optimized the performance of a novel magnetic cell sorting program (Depletion 3.1, CliniMACS System), for large-scale direct depletion of T-cells from peripheral blood progenitor cells (PBPC). The optimized D3.1 program can be utilized for large-scale, time saving direct T-cell depletion with excellent recovery of CD34+ cells and an effective reduction of T-cell numbers. Furthermore, a novel micro-chip based acoustophoresis technique was invesigated (paper IV) for removal of platelets from PBPC products and was found to efficiently deplete PBPC samples of intact platelets, whilst preserving the target leukocyte fraction, cell viability and progenitor cell colony-forming ability. Acoustophoresis is, thus, an interesting technology to improve current cell processing methods.
In summary, this work has provided knowledge on differentiation-associated changes in human myeloid development. Surface restriction markers for erythroid (group A antigen) and neutrophil differentiation (CD15) have been established and utilized to investigate the origin of reappearing host cells post-HSCT. In a clinical context, the level of A/B-antigen found to be acquired by donor group O RBC following transfusion or HSCT suggests implications for plasma component selection. Moreover, current clinical cell separation methodology was improved and the novel acoustophoresis technology was demonstrated to provide efficient platelet depletion, with a potential application in future clinical graft engineering.